Composition for inhibition of transplant rejection containing the phellinus linteus mycellia extract as an active ingredient

ABSTRACT

Disclosed is a composition for the inhibition of transplant rejection and the prevention and treatment of skin diseases, comprising a  Phellinus  sp. mycelial extract as an active ingredient. The  Phellinus  sp. mycelial extract significantly suppresses the production of antibodies to transplants without side effects, such as weight change. Based on natural material, the composition is non-toxic and harmless to the human body and thus can be used as an immunosuppressant for organ transplantation. Also, it stops oozing from sores and is applicable to the prevention and treatment of skin diseases, including atopy, allergic reactions, decubitus ulcers, and smallpox.

TECHNICAL FIELD

The present invention relates to a composition for inhibition oftransplant rejection, comprising a Phellinus sp. mycelial extract as anactive ingredient.

BACKGROUND ART

Transplant rejection occurs when the immune system of the recipient of atransplant attacks the transplanted organ or tissue. Thus, effectivesuppression of the immune response is known as a main factor determiningthe success of transplantation. In this regard, the development ofimmunosuppressive medications has brought about exceptional advances inthe transplantation of organs and tissues and the treatment ofautoimmune diseases and has made a great contribution to the study ofthe in vivo mechanism of immune responses to the transplanted organ ortissue.

As described, immunosuppressive drugs were developed to inhibit orattenuate transplant rejection. An example is cyclosporine A (U.S. Pat.No. 4,117,118) produced from Tolypocladium inflatum, a soil fungus.These immunosuppressive drugs not only help realize clinicallysuccessful organ transplantation, but also suggest the therapeutic usethereof in treating autoimmune diseases. Even though they are requiredto act selectively and specifically for T-cells only, conventionalimmunosuppressive drugs have an influence on a wide range of cellularfunctions including general signal pathways, causing side effects onother organs, which are healthy (see. S.-H. Lee et al., Korean J.Immunology, 19:375˜389 (1997)). For instance, cyclosporine A is known toshow side effects of chronic liver diseases and hypertension after hearttransplantation (see: J. E. F. Reynolds, et al., Martindale The ExtraPharmacopoeia, 31^(st) ed., pp. 557-562, Royal Pharmaceutical Society,London, 1996). Many attempts have been made to develop novelimmunosuppressive drugs free from side effects. FK-506 has recently beendiscovered to be an immunosuppressant, and has been commercialized.However, side effects of this drug have also been found (Clin.Transplantation, 11: 237-242 (1997)).

Mushrooms of Phellinus spp. are white-rot fungi belonging to thePhellinaceae family of the Aphylloporales order in the Basidiomycetesclass, and have ligneous fruiting bodies. These mushrooms are very rareperennial fungi that parasitize broadleaf trees, including mulberrytrees, wild mulberry trees, black oak trees, chestnut trees, oak trees,aspens, willow trees, etc. In ancient Korean and Chinese herb medicinebooks, the mushrooms of Phellinus spp. are described as various nameswith excellent medicinal effects. These mushrooms were known amongdealers of herbal medicines as legendary medicines from old timesbecause they were difficult to obtain. In recent times, these mushroomshave been identified as enhancing immunity upon chemotherapy for variouscancers on the digestive system, including stomach cancer, esophagealcancer, duodenal cancer, colon cancer, rectal cancer, and liver cancer,after resection. Further, they are known to have therapeutic activity onmetrorrhagia and leucorrhea, menstrual irregularity, andenterohemorrhage, and activation and detoxication effects on the fiveviscera and the stomach. However, nowhere has the use of Phellinus spp.as an immunosuppressant been disclosed in the prior art.

Leading to the present invention, intensive and thorough research on animmunosuppressant entailing no side effects, conducted by the presentinventors, resulted in the finding that an extract from Phellinus sp.mycelia significantly inhibits the immune response to transplantedorgans or tissues.

DISCLOSURE Technical Problem

It is therefore an object of the present invention to provide animmunosuppressive composition, useful in the prevention of transplantrejection, comprising a Phellinus sp. mycelial extract as an activeingredient.

It is another object of the present invention to provide a compositionfor the prevention and treatment of skin diseases, comprising aPhellinus sp. mycelial extract as an active ingredient.

Technical Solution

In order to accomplish the objects of the present invention, animmunosuppressive composition comprising a mycelial extract fromPhellinus sp. as an active ingredient is provided for the inhibition oftransplant injection.

Also, a composition comprising a Phellinus sp. mycelial extract as anactive ingredient is provided for the prevention and treatment of skindiseases.

ADVANTAGEOUS EFFECTS

The Phellinus sp. mycelial extract was found to significantly suppressthe production of antibodies to transplants without side effects, suchas weight change. Based on a natural material, the composition isnon-toxic and harmless to the human body, and thus can be used as animmunosuppressant for organ transplantation. Also, it arrests oozingfrom sores and is applicable to the prevention and treatment of skindiseases, including atopy, allergic reactions, decubitus ulcers, andsmallpox.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the levels of antibodies produced againstsplenocytes transplanted into mice administered with the Phellinuslinteus mycelial extract of the present invention or with saline (▪:control, Δ: experimental group).

FIG. 2 is a graph showing changes in the weight of the mice administeredwith the Phellinus linteus mycelial extract of the present invention andwith saline (▪: control, Δ: experimental group).

BEST MODE

In accordance with an aspect of the present invention, animmunosuppressive composition based on a Phellinus sp. mycelial extractis provided for the inhibition of transplant rejection.

The Phellinus sp. mycelial extract according to the present inventionwas tested for immunosuppressive effect on smallpox mouse models. Afterthe transplantation of splenocytes thereinto, the animal models wereadministered with the Phellinus sp. mycelial extract. ELISA analysis onthe animal models for the quantification of antibodies to thesplenocytes indicated that the Phellinus sp. mycelial extract of thepresent invention significantly inhibits the production of theantibodies (see FIG. 1). Therefore, the Phellinus sp. mycelial extractaccording to the present invention can be used as an immunosuppressantfor the prevention of transplant rejection.

Also, observations were made of whether the Phellinus sp. mycelialextract causes side effects in vivo. Almost no changes were found in theweight of the mice after the transplantation of splenocytes (see FIG.2). Hence, the Phellinus sp. mycelial extract can be used as animmunosuppressant for the prevention of transplant rejection without theoccurrence of side effects, such as changes in weight, upon organtransplantation.

In accordance with another aspect thereof, the present inventionprovides a composition for the prevention and treatment of skindiseases, comprising a Phellinus sp. mycelial extract as an activeingredient.

The Phellinus sp. mycelial extract according to the present inventionwas tested for therapeutic activity for skin diseases on smallpox mousemodels. After the administration of the Phellinus sp. mycelial extractthereinto, the smallpox mouse models were observed to stop oozing fromthe smallpox sores. Hence, the Phellinus sp. mycelial extract accordingto the present invention can be used for the treatment of skin diseases,such as atopy, allergic reactions, decubitus ulcers, smallpox, etc.

The preparation of the Phellinus sp. mycelial extract for the preventionof transplant rejection and the prevention and treatment of skindiseases in accordance with the present invention can be achieved by,but is not limited to, the methods disclosed in Korean Patent Nos.197446, 174433, and 124853.

Examples of the Phellinus sp. useful in the present invention includePhellinus linteus, Phellinus baumii, and Phellinus igniarius.

When used as medications, the composition may further comprise one ormore active ingredients having a function similar to that of thePhellinus sp. mycelial extract.

The Phellinus sp. mycelial extract in accordance with the presentinvention can be administered orally or non-orally, and may be providedin general medicinal forms. For clinical practice, the Phellinus sp.mycelial extract of the present invention may be used in oral ornon-oral forms. It is usually formulated in combination with a diluentor excipient, such as a filler, a thickening agent, a binder, a wettingagent, a disintegrant, a surfactant, etc. Solid agents intended for oraladministration of the extract of the present invention may be in theform of tablets, pills, powders, granules, capsules, and the like. Inthese solid agents, the Phellinus sp. mycelial extract of the presentinvention is formulated in combination with at least one excipient, suchas dextrin, starch, calcium carbonate, sucrose, lactose, or gelatine. Inaddition, a lubricant, such as magnesium stearate, talc, or the like,may also be added. Liquid agents intended for oral administrationinclude suspensions, internal use solutions, emulsion, syrups, and thelike. In addition to a simple diluent, such as water or liquid paraffin,various excipients, such as wetting agents, sweetening agents,aromatics, preservatives, and the like, may be contained in the liquidagents for the oral administration of the extract of the presentinvention. Also, non-oral dosage forms of the extract of the presentinvention include sterile aqueous solutions, non-aqueous solutions,suspensions and emulsions, freeze-dried agents, and suppositories.Non-aqueous solutions and suspensions made from propylene glycol,polyethylene glycol, vegetable oils, such as olive oil, and injectableesters such as ethyl oleate may be used. The basic materials ofsuppositories include Witepsol, macrogol, Tween 61, cacao butter,laurin, glycerol, and gelatin.

The effective dosage of the Phellinus sp. mycelial extract in accordancewith the present invention depends on various factors, including thepatient's weight, age, gender, state of health, diet, the time ofadministration, route of administration, excretion rate, etc. For oraladministration, the Phellinus sp. mycelial extract in accordance withthe present invention may be administered at a dose ranging from 550 to2,200 mg/day.

For application to the treatment of skin diseases, the Phellinus sp.mycelial extract of the present invention may be used alone or incombination with other therapies, including surgery, radiotherapy,hormonal therapy, chemical therapy and/or biological reactionregulators.

MODE FOR INVENTION

A better understanding of the present invention may be obtained in lightof the following examples, which are set forth to illustrate, but arenot to be construed to limit the present invention.

Example Immunosuppression Assay

An immunosuppression test was conducted with the Phellinus sp. mycelialextract of the present invention on smallpox mouse models (obtained fromMicrobiology Immunology Lab of the Medical College in Keio Univ.).

From two days before the transplantation of splenocytes (Dsg3−/−), fivesmallpox mice were administered orally with a Phellinus sp. mycelialextract powder (Mesima®; Han Kook Sin Yak) at a dose of 10 mg/kg/day.For 35 days (5 weeks) after the splenocyte transplantation, thePhellinus sp. mycelial extract was orally administered at a dose of 10mg/kg/day. For a control, physiological saline was used instead of theextract.

Blood samples were taken from the mice 7 days (1 week), 14 days (2weeks), 21 days (3 weeks), 28 days (4 weeks) and 35 days (5 days) afterthe transplantation, and were analyzed for the level of antibodies tothe splenocytes using ELISA. Throughout the experiment, the weights ofthe mice were measured every day.

The results are depicted in FIGS. 1 and 2.

In FIG. 1, the levels of the antibodies to the splenocytes are plottedagainst time for the experimental group and the control group. FIG. 2shows changes in weight for the experimental group and the controlgroup.

As seen in FIG. 1, the level of antibodies to the transplantedsplenocytes in the mice administered with the Phellinus sp. mycelialextract of the present invention was almost zero. From this, it isapparent that the Phellinus sp. mycelial extract according to thepresent invention effectively inhibits the production of antibodies totransplants.

In FIG. 2, it can be observed that the weight of the controladministered with physiological saline slightly increased immediatelyafter the transplantation, but sharply decreased from 7 days after thetransplantation, as transplant rejection occurred. In contrast, almostno change was found in the weight of the mice administered with thePhellinus sp. mycelial extract of the present invention. Demonstrated toeffectively inhibit the transplant rejection and cause no side effects,such as weight gain, the composition comprising the Phellinus sp.mycelial extract according to the present invention can therefore beused as an immunosuppressive medication applicable for organ or tissuetransplantation.

Furthermore, while the control suffered from sores due to smallpox, skindisease was suppressed in the mice administered with the Phellinus sp.mycelial extract of the present invention. Hence, the Phellinus sp.mycelial extract according to the present invention can be applied tothe treatment of skin diseases including atopy, allergic reactions,decubitus ulcers, smallpox, etc.

The composition of the present invention can be prepared as describedbelow.

Preparation Example 1 Preparation of Pharmaceutical Formulations

1-1. Preparation of Powder

Phellinus sp. mycelial extract   1 g Dextrin 0.1 g

The above ingredients were mixed and loaded into an airtight sac toproduce powder.

1-2. Preparation of Tablet

Phellinus sp. mycelial extract 500 mg  Dextrin 45 mg Mg Stearate  5 mg

These ingredients were mixed and prepared into tablets using a typicaltabletting method.

1-3. Preparation of Capsule

Phellinus sp. mycelial extract 500 mg Dextrin  50 mg

These ingredients were mixed and loaded into gelatin capsules accordingto a typical method to produce capsules.

1-4. Preparation of Injection

Phellinus sp. mycelial extract 50 mg/ml Diluted HCl BP added to form pH3.5 NaCl BP injection up to 1 ml

The Phellinus sp. mycelial extract was dissolved in a suitable volume ofan NaCl BP injection, and the solution was adjusted to a pH of 3.5 withdiluted HCl BP and to a desired volume with NaCl BP injection, followedby sufficient mixing. The solution was loaded into transparent 5 ml typeI ampules, which were hermetically sealed by melting, followed byautoclaving at 120° C. for 15 min to prepare injections.

1. An immunosuppressive composition for inhibition of transplantinjection, comprising a mycelial extract from Phellinus sp. as an activeingredient.
 2. The immunosuppressive composition according to claim 1,wherein the Phellinus sp. is Phellinus linteus, Phellinus baumii orPhellinus igniarius.
 3. The immunosuppressive composition according toclaim 1, wherein the composition is in a dosage form suitable for oral,intravenous or abdominal administration.
 4. The immunosuppressivecomposition according to claim 3, wherein the composition is orallyadministered at a dose from 550 to 2,200 mg/day.
 5. Theimmunosuppressive composition according to claim 1, wherein thecomposition suppresses production of antibodies after organ or tissuetransplantation, said antibodies causing transplant rejection.
 6. Acomposition for prevention and treatment of skin diseases, comprising aPhellinus sp. mycelial extract as an active ingredient.
 7. Thecomposition according to claim 6, wherein the skin diseases are atopy,decubitus ulcer, and/or smallpox.